Optical coherence photoacoustic microscopy

ABSTRACT

A system and method for providing an optical coherence photoacoustic (OC-PAM) microscopy. An OC-PAM microscope includes a light source that outputs light, a scanner, a detector, a transducer, and an image processing module. The scanner receives the light and scans the light across a sample. The detector receives reflected light from the sample in response to the scanned light. The transducer detects photoacoustic waves induced in the sample by the scanned light. The image processing module receives output from the detector and the transducer and generates a photoacoustic microscopy (PAM) image and an optical coherence tomography (OCT) image based on the received output from the detector and the transducer. The PAM and OCT image data may be fused to form a single, OC-PAM image. Additionally, a series of PAM images and OCT images, respectively, may be combined to generate three-dimensional PAM and OCT images, respectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit as a continuation of andpriority to U.S. patent application Ser. No. 13/524,813, filed on Jun.15, 2012, for “OPTICAL COHERENCE PHOTOACOUSTIC MICROSCOPY”, which claimspriority of U.S. Provisional application No. 61/497,323, filed on Jun.15, 2011, for “OPTICAL COHERENCE PHOTOACOUSTIC MICROSCOPY” each of whichare incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH FOR DEVELOPMENT

This invention was made with government support under 1 RC4 EY021357(Subcontract No. 2791031 Board of Regents of the University of WisconsinSystems) and 7 R21 EB008800-02 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

The present invention relates to optical coherence tomography (OCT) andphotoacoustic microscopy (PAM).

SUMMARY

OCT and PAM are two microscopic three-dimensional non-invasive imagingmodalities that are based on different contrast mechanisms. OCT is alow-coherent interferometer-based optical imaging modality that providesimaging of mainly the scattering properties of biological tissues. Byusing a broadband light source, OCT resolves the depth of a scattererthrough coherence gating.

In contrast, PAM is an optical-absorption based imaging modality thatdetects laser-induced ultrasonic waves as a result of specific opticalabsorption. When short laser pulses irradiate biological tissues,optical energy is absorbed by substances like hemoglobin and melanin andconverted to heat. Thermo-elastic expansions then occur, which lead tothe generation of wideband ultrasonic waves. The ultrasonic waves aredetected by an ultrasonic transducer and used to quantify the opticalabsorption properties of the sample. The waves may be used to form animage of the sample based upon the optical absorption contrast ofelements of the sample, such as tissue of a biological sample.

Due to the different contrast mechanisms, OCT and PAM can providedifferent, but complementary, information of biological tissues. OCTimages the microanatomy of a sample, such as a histology-likecross-sectional image of a retina. OCT can also measure blood flowvelocity by measuring the Doppler effect impinged on the probing light.In contrast, PAM images a microvasculature and blood oxygenation byusing multiple wavelength illumination.

Previously, light sources used in OCT and PAM were different. OCTgenerally uses near infrared, broadband and continuous light, such asproduced by a superluminescent diode (SLD), or infrared, virtuallycontinuous light, such as produced by a Ti:Sapphire laser withapproximately 80 MHz pulse repetition rate. In contrast, PAM generallyuses narrow band and pulsed lasers in the visible light spectrumtargeting, for instance, the absorption of hemoglobin. Use of nearinfrared (NIR) light in OCT allows for deeper penetration depth thanotherwise achievable using light in the visible spectrum. The selectionof light wavelength in PAM, including whether visible light or NIRlight, depends on the absorption spectrum of the targeted molecules.Additional information related to OCT and PAM imaging may be found inU.S. Pat. No. 8,025,406, the entire contents of which are herebyincorporated by reference.

Embodiments of the invention relate to systems and methods for opticalcoherence photoacoustic microscopy (OC-PAM), a multi-modal microscopicimaging modality that can simultaneously image the absorption andscattering contrasts of biological tissues non-invasively. OC-PAM usesone light source, such as a pulsed broadband laser or a swept laser thatoutputs pulsed swept laser light of a plurality of wavelengths in ashort scan period (e.g., less than 10 nanoseconds (ns)), tosimultaneously achieve both PAM functions, by detecting theabsorption-induced photoacoustic waves, and OCT functions, by detectingthe reflected light using an interferometer. In OC-PAM imaging, witheach laser pulse, an A-scan is generated for both OCT and PAM,respectively. Additionally, OC-PAM imaging generates inherentlyregistered PAM and OCT images, providing an ability to study thescattering and absorption of biological tissues.

Embodiments of the invention can be used for OC-PAM imaging of abiological sample, such as a human eye. For example, light of the OC-PAMmicroscopes disclosed herein may enter through a pupil and be directedto a retinal region of interest within an eye. Additionally, the systemsand methods disclosed herein may be used to image various biologicalsamples such as cells and molecules in suspension, physiologicalappendages, small animal organs (e.g., ears, skin, eyes, brain, internalorgans, etc.) and human eyes and skin.

In another embodiment, the invention provides an optical coherencephotoacoustic microscope including a light source that outputs light, asample, a detector, a transducer, and an image processing module. Thesample receives the light, which is scanned across the sample. Thedetector receives reflected fight from the sample in response to thescanned light. The transducer is positioned to detect photoacousticwaves induced in the sample by the scanned light. The image processingmodule receives output from the transducer and the detector andgenerates a photoacoustic microscopy (PAM) image and an opticalcoherence tomography (OCT) image based on the received output from thedetector and the transducer.

In another embodiment, the invention provides a method for opticalcoherence photoacoustic microscope. The method includes emitting lightfrom a light source and scanning the light across a sample. A detectorreceives reflected light from the sample in response to the scannedlight. The method further includes detecting, by a transducer,photoacoustic waves induced in the sample by the scanned light, andreceiving, by an image processing module, output from the detector andthe transducer. The image processing module generates a photoacousticmicroscopy (PAM) image and an optical coherence tomography (OCT) imagebased on the received output from the detector and the transducer.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an optical coherence photoacoustic microscopy(OC-PAM) microscope in a transmission mode according to embodiments ofthe invention.

FIGS. 2A-D illustrate light passing through the beam splitter 150 atvarious stages of OC-PAM.

FIGS. 3A-B illustrate examples of optical coherence tomography (OCT)image data.

FIG. 4 A illustrates an OCT B-scan of a mouse ear generated with theOC-PAM microscope of FIG. 1.

FIG. 4B illustrates a photoacoustic microscopy (PAM) B-scansimultaneously generated with the OC-PAM microscope of FIG. 1.

FIG. 4C illustrates a fused PAM B-scan and OCT B-scan generated with theOC-PAM microscope of FIG. 1.

FIG. 5 illustrates a maximum amplitude projection (MAP) image formedfrom PAM B-scans generated with the OC-PAM microscope of FIG. 1.

FIG. 6 illustrates an OC-PAM method for generating OCT and PAM imagedata using a single light source.

FIG. 7 illustrates an OC-PAM microscope in a reaction mode according toembodiments of the invention.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the following drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways. Also, it is to be understood thatthe phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limited. The use of“including,” “comprising,” or “having” and variations thereof herein ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items. The terms “mounted,”“connected,” and“coupled” are used broadly and encompass both direct and indirectmounting, connecting and coupling. Further, “connected” and “coupled”are not restricted to physical or mechanical connections or couplings,and can include electrical connections or couplings, whether direct orindirect. Also, electronic communications and notifications may beperformed using any known means including direct connections, wirelessconnections, etc.

It should be noted that a plurality of hardware and software baseddevices, as well as a plurality of different structural components maybe utilized to implement the invention. Furthermore, and as described insubsequent paragraphs, the specific configurations illustrated in thedrawings are intended to exemplify embodiments of the invention and thatother alternative configurations are possible.

FIG. 1 illustrates an optical coherence photoacoustic microscopy(OC-PAM) microscope 100 according to embodiments of the invention. Themicroscope 100 includes a controller 105 for providing a user interfacefor operating and viewing images generated by the microscope 100. Thecontroller 105 may be implemented by hardware, software, or acombination thereof. For instance, the controller 105 may include one ormore of a general purpose processing unit, a digital signal processor, afield programmable gate array (FPGA), an application specific integratedcircuit (ASIC), and other processing devices operable to carry out thefunctions attributable to the controller 105 described herein. Thecontroller 105 may further include a memory, or be coupled to a memory,that stores instructions executed by the controller 105 to carry out theaforementioned functions, may store data for the controller 105, such asimage data, and may load data to the controller 105, such as programdata, calibration data, etc. for use by the controller 105 duringoperation of the microscope 100. Additionally, the controller 105 mayinclude user interface components, such as a display, a graphical userinterface (GUI), keyboard, mouse, touch screen, etc., to allow a user tocontrol and interact with the microscope 100, and to view resultingimages.

The controller 105 is coupled to and provides signals to a digital delaygenerator 110. In response to the signals from the controller 105 and aclock output, such as a clock of an analog output, the digital delaygenerator 110 triggers a laser 115 to output pulses. The digital delaygenerator 110 is also coupled to a charge coupled device (CCD) camera120 to trigger image capture by the CCD camera 120 with appropriatetiming. In some embodiments, a complementary metal-oxide-semiconductorCMOS camera is used in place of the CCD camera 120.

The laser 115 includes a broadband dye laser 125 pumped by a pump laser130. The pump laser 130 is a frequency-double Q-switched Nd:YAG(neodymium-doped yttrium aluminum garnet) laser. For instance, the pumplaser 130 may be a PSOT-10-100-532 laser sold by Elforlight Ltd., whichproduces a 532 nm, 10 μJ/pulse pulse with a 2 ns pulse duration, 30 kHzpulse repetition rate. The output light of the laser 115 has a centerwavelength of 580 nm and a bandwidth of 20 nm with, for instance, a 5kHz pulse repetition rate. The particular laser output may be varieddepending on the application. Generally, as the center wavelength of thelaser output increases, the bandwidth also increases. For example, whenthe laser output has a center wavelength of 830 nm, the bandwidth may be50 nm (i.e., 830 nm+/−25 nm); and when the laser output has a centerwavelength of 1000 nm, the bandwidth may be 100 nm (i.e., 830 nm+/−50nm). In general, the square of the center wavelength divided by thebandwidth

$\frac{830^{2}}{50} = {\frac{688900}{50} = {13\text{,}778.}}$is within an approximate range of about 10,000 to 20,000, although, incertain embodiments, the ratio may be higher than 20,000 or lower than10,000. For example,

$( {{i.e.},\frac{{CenterWavelength}^{2}}{Bandwidth}} )$In some instances, the spectrum of the laser pulses from the laser 115has a relatively high noise level, which may reduce the quality of theOCT images acquired by the microscope 100. However, this reduction inquality may be somewhat offset by using pulsed light with stablespectral performance.

In some embodiments, the laser 115 generates output using componentsdifferent than the pump laser 130 and dye laser 125. Furthermore, insome embodiments, the laser 115 is a swept laser source that outputspulsed swept laser light of a plurality of wavelengths, which are sweptin a scan period shorter than 10 ns. Alternatively, the laser 115 may bea pulsed supercontinuum light source, a pulsed broadbandsuperluminescent diode (SLD), or a broadband Ti:Sapphire laser.Additionally, in some embodiments, the light spectrum emitted by thelaser 115 may tend closer to or include near infrared (NIR) wavelengthsto achieve better imaging depth and ophthalmic applications. Theparticular wavelengths emitted by the laser 115 may vary depending onthe targeted absorber in a sample to be imaged.

The light output by the laser 115 is focused by a lens 135 on a singlemode optical fiber (SMF) 140. The SMF 140 outputs the light towards alens 145, which collimates the light and directs it to a beam-splittercube 150 via a mirror 155 a. The light received by the beam-splittercube 150 is split into sample arm light 160 and reference arm light 165,which is more clearly illustrated in FIGS. 2A-B. The sample arm light160 is directed by an x-y scanner 170, under control of the controller105, towards a lens 135 c. The lens 135 c focuses the sample arm light160 onto a sample 180.

In response to the sample arm light 160, the sample 180 (a) reflects aportion of the light and (b) absorbs a portion of the light. Theabsorbed portion of light is converted to heat and causes thermo-elasticexpansions to occur in the sample 180. The thermo-elastic expansionsgenerate wideband ultrasonic waves, which are detected by an ultrasonictransducer 185. The ultrasonic transducer 185 is a needle ultrasonictransducer (30 MHz; bandwidth: 50%; active element diameter: 0.4 mm),which is inserted into a plastic tube 190 filled with ultrasonic gel.The tube 190 and transducer 185 are placed under and in physical contactwith the sample 180. The transducer 185 outputs an analog signal to anamplifier 192, which outputs the signal, amplified, to a digitizer 193.The digitizer 193 is coupled to the controller 105 to provide thecontroller 105 with the digitized, amplified signal from the transducer185. The distance between the ultrasonic transducer 185 and sample 180in the application illustrated in FIG. 1 is approximately 6 mm. In otherembodiments, alternate transducers configurations are used in place ofthe transducer 185 and/or tube 190, and may be positioned in alternatelocations with respect to the sample 180, such as above the sample 180.In some embodiments, one or more of the ultrasonic transducer 185,amplifier 192, and digitizer 193 are combined into an integrated unit.In some embodiments, the transducer 185 is integrated into a contactlens for placement directly on the eye to be scanned.

After the beam splitter 150 splits the light into the sample arm 160 andthe reference arm light 165, the reference arm light 165 passes throughan iris 195 and is reflected by a mirror 155 b towards a mirror 155 c.The reference arm light 165 reaches a glass plate 200, which allows themajority of the reference arm light 165 to pass through to the mirror155 c, but reflects a portion of the reference arm light 165 towards aphoto diode 205. The photo diode 205 outputs a signal to the controller105 indicating the receipt of the reflected reference arm light 165. Thesignal from the photo diode 205 triggers capture by the controller 105of the digitized, amplified transducer data emitted by the digitizer193.

The glass plate 200 is a BK7 glass plate, which is used to compensatefor the group-velocity dispersion mismatch between the sample arm 160and the reference arm 165. In some embodiments, a different glass plateis used in the microscope 100.

The reference arm light 165 that passes through the glass plate 200 isreflected by the mirror 155 c back towards the glass plate 200 andmirror 155 b. The majority of the reflected light is passed through theglass plate 200 and proceeds to the mirror 155 b, which reflects thereference arm light 165 through the iris 195 on route to the beamsplitter 150. Simultaneously, the light reflected by the sample 180passes back through the leas 135 c and is reflected by the x-y scanner170 towards the beam splitter 150. The returning sample arm light 160then passes through the beam splitter 150 while the returning referencearm light 165 is reflected by the beam splitter 150, resulting in thereturning sample aim light 160 and reference arm light 165 beingcombined by the beam splitter 150. FIGS. 2C-D illustrate the combinationof the sample arm light 160 and reference arm light 160 by the beamsplitter 150. In some embodiments, in place of the beam splitter 150,one or more fiber couplers may be used to split and combine the samplearm light 160 and reference arm light 165. The combined light is thenfocused by a lens 135 d on a single mode fiber 215. The single modeFiber 215 is a fiber optic medium that transmits the light to a detector220 that includes a spectrometer 225 and the CCD camera 120. Thecombined sample arm light 160 and reference arm light 165 provide aninterference pattern, as each arm has traveled approximately the sameoptical distance.

The spectrometer 225 includes a diffractive grating, such as atransmission grating with 1800 line pairs per millimeter (lp/mm) and animaging lens (e.g., having f=150 mm). The diffractive grating dispersesthe light from the single mode fiber 215 as a line spectrum on theimaging leas, which focuses the line spectrum on the CCD camera 120. TheCCD camera 120 is a line scan type, such as an Avitva-SM2-CL-2010, with2048 pixels operating in 12-bit mode, e2V. As previously noted, thedigital delay generator 110 provides a triggering signal to triggerimage capture by the CCD camera 120 when the dispensed light reaches theCCD camera 120. The exposure time of the camera is based on the pulsewidth of the light emitted by the laser 115 and is, generally,approximately the same time length as the period of the pulse width ofthe light. The shutter may be open for more or less time than the periodof the pulse width of the laser 115. The effective exposure time is thetime that the laser is emitting light and the shutter of the camera 120is open.

The microscope 100 scans an area (x by y) of the sample 180, one (x,y)coordinate point at a time. The image data captured by the CCD camera120 form the OCT images, while the transducer data obtained by thetransducer 185 form the PAM information.

With respect to OCT imaging, for each scanned point on the sample 180,the CCD camera 120 captures one line of image data on the CCD camera 120for each coordinate point (an “A-scan”). FIGS. 3A-B illustrate low-passfiltered image data obtained by the CCD camera 120 based on theillumination of a single point on the sample 180. FIG. 3A illustratesthe normalized intensity for the light received by the CCD camera 120 atthe various wavelengths of the light as dispersed by the spectrometer225. FIG. 3B illustrates a calculated point-spread-function (PSF) of theOCT data. In the example illustrated in FIGS. 3A-B the sample 180 was amirror, the path length difference was set to 0.5 mm, and the exposuretime of the CCD camera 120 was set to 10 μs. As shown in FIG. 3B, themeasured depth resolution is 9.5 μm in air.

A cross-sectional tomograph (B-scan) may be achieved by laterallycombining a series of the A-scans. Thus, after scanning the x by y areaof the sample 180, three-dimensions of image data have been capturedcorresponding to the x-dimension of the sample 180, the y-dimension ofthe sample 180, and the z-dimension (depth) of the sample 180. Thez-dimension is based on the spectral information, including theintensity of the various wavelengths of light received by the CCD camera120.

FIG. 4A illustrates a B-scan OCT image 250 generated by the OCT imagingcomponents of the microscope 100. The OCT image 250 shows across-section of a portion of the mouse ear. In the OCT image 250, thedepth resolution is sufficient to resolve different anatomical featuresincluding the epidermis 255, dermis 260, and cartilaginous backbone 265of the sample 180, which is a mouse ear. In this example, the OCT imagereaches about half of the thickness of the mouse ear.

With respect to PAM imaging, for each scanned point on the sample 180,the transducer 185 captures ultrasound data over time to generate anA-scan image which indicates the depth of various components of thesample 180. The series of A-scan images are laterally combined to form aB-scan image having three-dimensions of ultrasound data corresponding tothe x-dimension of the sample 180, the y-dimension of the sample 180,and the z-dimension (depth) of the sample 180. The z-dimension data isbased on the timing of the ultrasonic waves received by the transducer.

FIG. 4B illustrates a B-scan PAM image 270 generated by the PAM imagingcomponents of the microscope 100. The PAM image 270 shows a crosssection of a portion of a mouse ear, with the cross section of bloodvessels 275 of the mouse ear appearing as clusters of high amplitudeultrasonic signals. However, information about the anatomy of the tissueis not present where no significant absorption of the sample arm light160 occured.

For example, to generate the B-scan image data for either of the B-scanOCT image 250 or the B-scan PAM image 270, while the sample arm light160 is scanned in the one dimension (e.g., x=0 to n), the otherdimension is fixed (e.g., y=0). For each x-position, an A-scan isgenerating, resulting in a vertical line of image data. To form theB-scan image 250 or 270, the series of vertical lines of image data arecombined. Since both the OCT image 250 and PAM image 270 are generatedfrom the same photons, they are inherently and precisely co-registeredin the lateral directions (e.g., the x- and y-dimensions), which aredetermined by the optical scanning. In the depth direction, however,registration of the two imaging modes is not automatic. One techniquefor image registration in the depth direction is to first establish arelationship of the two images in the depth direction by, for instance,imaging a flat absorbing surface such as black tape. The PAM image willthen be scaled and interpolated accordingly and then fused with the OCTimage.

FIG. 4C illustrates an example of a fused OCT and PAM image 280, whichincludes a combination of the OCT image 250 and PAM image 270. The fusedimage 280 is a cross sectional view of the scanned portion of the mouseear showing the blood vessels 275 of the PAM image 270 and the epidermis255, dermis 260, and cartilaginous backbone 265 of the OCT image 250.

FIG. 5 illustrates a maximum-amplitude-projection (MAP) 285 of a 3D PAMdataset generated by the microscope 100. A series of B-scans generatedby the PAM components of the microscope 100 are stacked to generate theMAP 285. The MAP 285 shows a top-down view of the sample 180, i.e., asif viewing the sample 180 from a position of the lens 135 c above thesample 180. As shown in FIG. 5, the B-scan 270 of FIG. 4B is a portionof the MAP 285 centered at the horizontal line 290. The B-scan 270,along with a series of additional B-scans, are stacked such that theblood vessels 275 of multiple B-scans combine to form the blood vessel290 shown in FIG. 5. The PAM dataset making up the MAP 285 includes256×256 PAM A-scans covering an area of 2.6×2.6 mm².

FIG. 6 illustrates a method 300 of generating OC-PAM images with asingle light source using, for instance, the microscope 100.Accordingly, the method 300 is described with reference to themicroscope 100; however, other microscopes using a single light, sourcemay also perform the steps of method 300. In step 302, the laser 115emits broadband pulsed light towards the beam splitter 150. In step 304,the beam splitter 150 splits the emitted broadband pulsed light into thesample arm light 160 and the reference arm light 165. In step 306, thex-y scanner 170 directs the sample arm light 160 to a point on thesample 180. A portion of the sample arm light 160 is absorbed by thesample 180 and converted into photoacoustic waves, and another portionof the sample arm light 160 is reflected by the sample 180 back towardthe beam splitter 150.

In step 308 a, the photoacoustic waves generated by the sample 180 arereceived by the transducer 185. Meanwhile, a portion of the referencearm light 165 is reflected off of the glass 200 and is received by thephotodiode 205 in step 310 a, which, in step 312 a, triggers thecontroller 105 to capture the PAM data received by the transducer 185.Essentially simultaneously with step 308 a, in step 308 b, the beamsplitter 150 combines the portion of the sample arm light 160 reflectedby the sample 180 with the reference arm light 165 returning frommirrors 155 b and 155 c, and provides the combined light to the detector220. In step 310 b, the detector 220 is triggered by the digital delaygenerator 110 and, in response, in step 312 b, the detector 220 capturesOCT image data generated by the combined light reaching the detector220. The detector 220 then provides the OCT image data to the controller105. In step 314, the OCT image data and PAM image data captured in step312 are received by an imaging module (not shown) of the controller 105for processing and may be saved in a memory of the controller 105.

In step 316, the controller 105 determines whether additional portionsof the sample 180 remain to be scanned. If additional portions remain,the controller 300 returns to step 302 and proceeds to generateadditional OCT and PAM data for the next point on the sample. Once allof the sample points of the sample 180 have been scanned as determinedin step 316, the controller 105 proceeds to step 338 for imageprocessing of the OCT and PAM data. For instance, images such as shownin FIG. 3A to 5 are generated by the imaging module of the controller105 and saved in a memory of the controller 105 or output by thecontroller 105 to a display tor viewing, to another computing device forfurther processing or display, to a remote device over a network, to aremote storage device on a network, or to another device.

FIG. 7 illustrates an OC-PAM microscope 400 having an alternatearrangement. The microscope 400 has several components similar to themicroscope 100 and such components are like-numbered. Light emitted fromthe laser 115 is filtered by spatial filter 405 and collimated beforereaching the beam splitter 150, which, like the microscope 100, splitsthe emitted light into the sample arm light 160 and reference arm light165. The microscope 400 functions with respect to the reference armlight 165 in a manner similar to the microscope 100. The sample armlight 160 is focused by the lens 135 c and scanned across the sample 180via the x-y scanning mirror 170. In the microscope 400, the transducer185 is positioned above the sample 180, rather than below the sample asin the microscope 100. Additionally, as an alternate to the tube 190 andultrasound gel of the microscope 100, the transducer 185 of themicroscope 400 is positioned in a water tank 410. Like the microscope100, the microscope 400 captures OCT data with the detector 220 and PAMdata with the transducer 185, which are generated based on the samephotons output by the laser 115 and are, therefore, inherently andprecisely co-registered in the lateral directions.

The microscopes 100 and 400 of FIGS. 1 and 7, respectively, include anx-y scanner 170 to scan the light across the sample 180. In someembodiments, however, the light from the laser 115 remains fixed whilethe sample 180, which is on a controlled scanning platform, is moved bythe platform to scan the light across the sample 180. For instance, inthese embodiments, the x-y scanner 170 may be replaced by a fixed mirroror may be held in a fixed position during a scan.

Thus, the invention provides, among other things, an OC-PAM system andmethod for simultaneously capturing OCT and PAM image data of a sampleinduced by a single light source.

In one embodiment, the invention provides an optical coherencephotoacoustic microscope. The microscope includes a light source thatoutputs broadband pulsed light, a scanner, a Michelson interferometerwith a spectrometer as a light detector, an ultrasonic transducer, andan image processing module. The scanner receives the light and scans thelight across a sample. The spectrometer in a detection arm of theinterferometer receives back-scattered light from the sample in responseto the scanned light, which interferes with reflected light from areference arm of the interferometer. The transducer detectsphotoacoustic waves induced in the sample by the scanned light in atransmission mode, where the transducer is placed on a side of thesample opposite the scanning light. The image processing module receivesoutput from the spectrometer and the ultrasonic transducer and generatesan optical coherence tomography (OCT) image and a photoacousticmicroscopy (PAM) image based on the received output from thespectrometer and the transducer.

In another embodiment, the invention provides a method for opticalcoherence photoacoustic microscopy. In the method, broadband pulsedlight is emitted from a light source and coupled into a source aim of aMichelson interferometer. The light is scanned across a sample. Aspectrometer in a detection arm of the interferometer receives acombination of light back-scattered from the sample in response to thescanned light and light reflected from a reference arm of theinterferometer. An ultrasonic transducer detects photoacoustic wavesinduced in the sample by the scanned light in a reflection mode, wherethe transducer is placed on a same side of the sample as the scanninglight. An image processing module receives output from the spectrometerand the transducer and generates a photoacoustic microscopy (PAM) imageand an optical coherence tomography (OCT) image based on the receivedoutput from the transducer and the spectrometer.

The systems and methods described herein may be used for the diagnosisand evaluation of age-related macular degeneration, geography atrophy,diabetic retinopathy, premature retinopathy, glaucoma, ocular tumors,retinal edema, retinal detachment, several types of ischemicretinopathy, and brain disorders. The systems and methods may further beused to monitor therapy on retinal diseases that use nano-particles andto provide therapy whereby the photons from the laser 115 are absorbedby the nano-particles to trigger a therapeutic reaction. Additionally,OC-PAM imaging may be used to diagnose diseases that may be diagnosedthrough morphology and functions of the retinal vessels, such as astroke and Alzheimer's disease. Various features and advantages of theinvention are set forth in the following claims.

What is claimed is:
 1. A method of optical coherence photoacousticmulti-modal microscopic imaging of a target, the method comprising:generating, substantially simultaneously using a light source, A-scansfor optical coherence tomography and photoacoustic microscopy of thetarget; forming, using a processor, B-scan images for optical coherencetomography and photoacoustic microscopy of the target co-registered in alateral direction and both formed from photons generated from theA-scans; and generating, using the processor, a fused image of thetarget from B-scan image data for optical coherence tomography andphotoacoustic microscopy, wherein the photoacoustic microscopy B-scanimage data is scaled and interpolated to be co-registered with theB-scan image for optical coherence tomography in a depth direction andto fuse with the optical coherence tomography B-scan data to form acombined optical coherence tomography and photoacoustic microscopyimage.
 2. The method of claim 1, wherein the light source is one of apulsed broadband laser that outputs pulsed broadband light and a sweptlaser source that outputs pulsed swept laser light of a plurality ofwavelengths, wherein the wavelengths are swept in a scan period shorterthan 10 nanoseconds.
 3. The method of claim 2, wherein the light sourceis the swept laser source and the detected photoacoustic waves haveenergy from the sum of the pulsed swept laser light of the plurality ofwavelengths in the scan period.
 4. The method of claim 1, furtherincluding moving the light source to a second point on the target toobtain additional A-scans for optical coherence tomography andphotoacoustic microscopy of the target.
 5. The method of claim 4,wherein moving the light source to a second point on the target includesscanning the light via an x-y scanner to direct light with respect tothe target.
 6. The method of claim 1, further including splitting, via abeam splitter, the light into a sample arm light and a reference armlight.
 7. The method of claim 6, further including: partially reflectingand partially transmitting the reference arm light; detecting, with aphotodiode, the reflected reference arm light; and outputting, based ondetecting the reflected light with the photodiode, a trigger signal toinitiate data acquisition for the photoacoustic microscopy A-scan. 8.The method of claim 7, further including combining the reflected light,before reaching the detector, with the partially transmitted referencearm light for the optical coherence tomography A-scan.
 9. An opticalcoherence photoacoustic microscopy controller apparatus comprising: amemory storing instructions for execution; and a computing deviceconfigured to execute the instructions stored in the memory to least:generate, substantially simultaneously using a light source, A-scans foroptical coherence tomography and photoacoustic microscopy of a target;form B-scan images for optical coherence tomography and photoacousticmicroscopy of the target co-registered in a lateral direction and bothformed from photons generated from the A scans; and generate a fusedimage of the target from B-scan image data for optical coherencetomography and photoacoustic microscopy, wherein the photoacousticmicroscopy B-scan image data is scaled and interpolated to beco-registered with the B-scan image for optical coherence tomography ina depth direction and to fuse with the optical coherence tomographyB-scan data to form a combined optical coherence tomography andphotoacoustic microscopy image.
 10. The apparatus of claim 9, whereinthe light source is one of a pulsed broadband laser that outputs pulsedbroadband light and a swept laser source that outputs pulsed swept laserlight of a plurality of wavelengths, wherein the wavelengths are sweptin a scan period shorter than 10 nanoseconds.
 11. The apparatus of claim10, wherein the light source is the swept laser source and the detectedphotoacoustic waves have energy from the sum of the pulsed swept laserlight of the plurality of wavelengths in the scan period.
 12. Theapparatus of claim 9, wherein the instructions, when executed, furtherconfigure the computing device to move the light source to a secondpoint on the target to obtain additional A-scans for optical coherencetomography and photoacoustic microscopy of the target.
 13. The apparatusof claim 12, wherein moving the light source to a second point on thetarget includes scanning the light via an x-y scanner to direct lightwith respect to the target.
 14. The apparatus of claim 9, wherein theinstructions, when executed, further configure the computing device tosplit, via a beam splitter, the light into a sample arm light and areference arm light.
 15. The apparatus of claim 14, wherein theinstructions, when executed, further configure the computing device to:partially reflect and partially transmit the reference arm light;detect, with a photodiode, the reflected reference arm light; and outputbased on detecting the reflected light with the photodiode, a triggersignal to initiate data acquisition for the photoacoustic microscopyA-scan.
 16. The apparatus of claim 15, wherein the instructions, whenexecuted, further configure the computing device to combine thereflected light, before reaching the detector, with the partiallytransmitted reference arm light for the optical coherence tomographyA-scan.